The DNA of Escherichia coli is largely confined to one or a few compact bodies known as nucleoids. Such compaction can cause dramatic changes in the reactivity of the DNA. Factors affecting nucleoid compaction have been difficult to characterize because of a lack of suitable experimental systems. In a test of the importance of macromolecular crowding by the surrounding cytoplasm on nucleoid compaction, we lysed cells under conditions that retained cell boundaries. Release of the cytoplasm allowed an immediate expansion of the DNA to fill the residual cell walls, in accord with a dominant role of crowding. However, if polylysine was added before lysis, the normal expansion of the DNA upon lysis was prevented; well defined, compact nucleoids persisted. Polylysine was used in a new procedure for nucleoid isolation that, for the first time, releases nucleoids that retain the general shape and state of compaction that was present in vivo. The procedure has been applied to exponential phase cells and cells treated with chloramphenicol or nalidixate to alter the morphology of their nucleoids. The notably unstable nucleoids of rifampicin-treated cells were obtained in compact, stable form with this procedure. Nucleoids released in the presence of polylysine were easily handled, and provided well-defined DNA fluorescence and phase contrast images. Uniform populations of nucleoids retaining characteristic shapes could be isolated after formaldehyde-fixation and heating with sodium dodecyl sulfate. Biochemical and structural characterizations of the released nucleoids are underway.